Novel light-activated compositions and methods using the same

ABSTRACT

The invention includes light-activated compositions and methods that are useful for promoting cell death or growth. In certain embodiments, the compositions comprise quantum dots (QD).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/559,577, filed Sep. 19, 2017, now allowed, which is a 35 U.S.C. §371 national phase application of, and claims priority to, InternationalApplication No. PCT/US2016/023191, filed Mar. 18, 2016, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.62/136,128, filed Mar. 20, 2015, all of which applications areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

The small size and tunability of nanomaterials has engendered greatinterest in biological applications such as diagnostics andtherapeutics. Silver and metal oxide nanoparticles show bactericidaleffects in a range of microorganisms. Metal nanoparticles may act asinfrared absorbers and induce cell death by heating the surroundingmedium. This toxic effect is generally attributed to the generation ofgeneric reactive oxidative species (ROS) and free radicals, which candamage biomolecules such as DNA, RNA and proteins.

Cells that grow in aerobic environments have mechanisms to mitigate oruse ROS through redox homeostasis processes. Within cell types, aspecific redox homeostasis is maintained by the cell, and this governsthe function of broad processes including metabolism and signaltransduction. The generation of the specific ROS species is determinedby the redox environment present in the cell. Perturbation outside of acells redox homeostasis is linked to cell death in Escherichia coli,cancer, cardiovascular disease, and ageing in humans, and irreversibletissue damage in plants.

Quantum dots (QDs) are nanoparticles made of semiconductor materials andsmall enough to exhibit quantum mechanical properties. Specifically, theQD's excitons are confined in all three spatial dimensions. Due toquantum confinement, QDs have quantized energy states that, whenphoto-excited, have excited electrons and holes available forinteractions.

The electronic properties of QDs are intermediate between those of bulksemiconductors and of discrete molecules. Electronic characteristics ofa QD are closely related to its size and shape. For example, the bandgap in a QD, which determines the frequency range of emitted light, isinversely related to its size. In fluorescent dye applications, thefrequency of emitted light increases as the size of the QD decreases.Consequently, the color of emitted light shifts from red to blue whenthe size of the quantum dot is made smaller. This allows the excitationand emission of QD to be highly tunable. Since the size of a QD may beset when it is made, its conductive properties may be carefully tunedand/or controlled. QD dot assemblies consisting of many different sizes,such as gradient multi-layer nanofilms, can thus exhibit a range ofdesirable emission properties.

There is a need in the art for novel compositions that can be used topromote growth and/or death of cells. In certain embodiments, thecompositions promote selective growth and/or death of a cell type in thepresence of another cell type. The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a composition comprising at least onesemiconductor-containing nanoparticle. The present invention furtherprovides a method of promoting growth, killing, or preventing and/orhampering growth, of a first cell. The present invention furtherprovides a method of altering redox homeostasis in a cell.

In certain embodiments, the at least one nanoparticle has a band edgeredox potential such that, under conditions whereby the at least onenanoparticle penetrates a first cell, irradiation of the compositionwith radiation ranging from about 400 nm to about 1,000 nm in thepresence of the first cell promotes growth, kills, or prevents and/orhampers growth, of the first cell.

In certain embodiments, the at least one nanoparticle comprises aquantum dot (QD).

In certain embodiments, the composition is irradiated with radiationranging from about 750 nm to about 1,000 nm.

In certain embodiments, the at least one nanoparticle comprises CdTe,and wherein the at least one nanoparticle is at least partially coatedwith at least one selected from the group consisting of ZnS and CdS.

In certain embodiments, the composition further comprises the firstcell. In other embodiments, the first cell is a bacterium. In yet otherembodiments, the bacterium comprises at least one selected from thegroup consisting of K. pneumonia, E. coli, S. aureus, P. aeruginosa, A.baumannii and S. typhimurium. In yet other embodiments, the first cellcomprises a gram-negative bacterium. In yet other embodiments, the firstcell comprises a multi-drug resistant gram-negative bacterium. In yetother embodiments, the composition further comprises at least onegram-negative antibacterial agent. In yet other embodiments, the atleast one antibacterial agent is a cephalosporin antibiotic,fluoroquinone, or protein synthesis inhibitor. In yet other embodiments,the at least one antibacterial agent is selected from the groupconsisting of Amikacin, Aztreonam, Cefdinir, Cefaclor, Cefamandole,Cefditoren, Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime,Cefprozil, Cefuroxime, Ceftazidime, Ceftibuten, Ceftobiprole,Ceftriaxone, Chloramphenicol, Ciprofloxacin, Clindamycin, Colistin,Ertapenem, Doripenem, Gatifloxacin, Gentamicin, Imipenem/Cilastatin,Kanamycin, Levofloxacin, Meropenem, Metronidazole, Moxifloxacin,Neomycin, Netilmicin, Ofloxacin, Paromomycin, Polymyxin B, Streptomycin,Thiamphenicol, Tigecycline, and Tobramycin.

In certain embodiments, the concentration or amount of the antibacterialagent in the composition that is required to kill, or prevent and/orhamper the growth of, gram-negative bacteria is lower than theconcentration or amount of the antibacterial agent that is required tokill, or prevent and/or hamper the growth of, gram-negative bacteriawhen the antibacterial agent is used in the absence of the at least onenanoparticle.

In certain embodiments, the composition further comprises a second cell,wherein irradiation of the composition has no measurable effect on thegrowth, metabolism and/or survival of the second cell. In otherembodiments, the second cell is mammalian.

In certain embodiments, irradiation of the composition in the presenceof the first cell promotes growth of the first cell, and wherein the QDcomprises CuInS₂.

In certain embodiments, irradiation of the composition in the presenceof the first cell kills, or prevents and/or hampers growth of, the firstcell, and wherein the QD comprises CdTe.

In certain embodiments, irradiation of the composition in the presenceof the first cell promotes growth of the first cell, and wherein theband edge of the conduction band state (reduction potential) of the QDis about +0.2 V and the band edge of the valence band state (oxidationpotential) of the QD is about −1.8 V, as referenced to NHE (standardhydrogen electrode).

In certain embodiments, irradiation of the composition in the presenceof the first cell promotes death or prevents and/or hampers growth ofthe first cell, and wherein the band edge of the conduction band state(reduction potential) of the QD is about +0.35 V and the band edge ofthe valence band state (oxidation potential) of the QD is about −2.1 V,as referenced to NHE (standard hydrogen electrode).

In certain embodiments, irradiation of the composition has at least oneeffect selected from the group consisting of: changing redox homeostasisin the first cell, and generating at least one light-activated reactivespecies in the first cell.

In certain embodiments, the method comprises irradiating the first cellwith radiation ranging from about 400 nm to about 1,000 nm in thepresence of at least one semiconductor-containing nanoparticle with aband edge redox potential, under conditions whereby the at least onenanoparticle penetrates the first cell. In other embodiments, the firstcell comprises a gram-negative bacterium and wherein the first cell isfurther contacted with at least one gram-negative antibacterial agent.

In certain embodiments, the first cell is in the presence of a secondcell, and wherein irradiation of the first and second cells in thepresence of the at least one nanoparticle has no measurable effect onthe growth, metabolism and/or survival of the second cell. In yet otherembodiments, the second cell is mammalian. In yet other embodiments,growth of the first cell is promoted, and wherein the QD comprisesCuInS₂. In yet other embodiments, growth of the first cell is preventedand/hampered, or killing of the first cell is promoted, and wherein theQD comprises CdTe. In yet other embodiments, growth of the first cell ispromoted, and wherein the band edge of the conduction band state(reduction potential) of the QD is about +0.2 V and the band edge of thevalence band state (oxidation potential) of the QD is about −1.8 V, asreferenced to NHE (standard hydrogen electrode). In yet otherembodiments, growth of the first cell is prevented and/or hampered, orkilling of the first cell is promoted, and wherein the band edge of theconduction band state (reduction potential) of the QD is about +0.35 Vand the band edge of the valence band state (oxidation potential) of theQD is about −2.1 V, as referenced to NHE (standard hydrogen electrode).In yet other embodiments, the at least one nanoparticle comprises CdTe,and wherein the at least one nanoparticle is at least partially coatedwith at least one selected from the group consisting of ZnS and CdS. Inyet other embodiments, irradiation has at least one effect selected fromthe group consisting of: changing redox homeostasis in the first cell,and generating at least one light-activated reactive species in thefirst cell.

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1A illustrates STS measurements of cadmium selenide (CdSe), cadmiumtelluride (CdTe), and copper indium sulfide (CuInS₂, CIS) nanoparticleswith bandgaps of ˜1.9 eV, demonstrating the different redox potentialsof the materials. FIG. 1B is a schematic illustration of the impact oncells from exposure to quantum dots. Below a concentration threshold,the quantum dots had no effect on cell health in dark. Upon lightexposure, the formation of light-activated reactive species (LARS) ledto phototoxicity for CdTe, photo-proliferation (enhanced growth) forCIS, and no effect for CdSe.

FIG. 2A comprises a series of graphs illustrating absorbancemeasurements of each material at various sizes. The scale bars in theinset histograms were color coded to the absorbance spectra, and were 50nm in all cases except the 2.4 eV CdTe, which was 25 nm. FIG. 2Bcomprises a graph illustrating STS measurements of individual quantumdots. Color coding matches FIG. 2A. FIG. 2C comprises a graphillustrating the average CB/VB position of various individual quantumdots for each material and size. The extent of the colored box indicatesthe average while the error bar indicates the upper limit of the bandposition.

FIG. 3A comprises a series of graphs illustrating optical density curvesused to track E. coli growth over time with exposure to QDs in light anddark. FIG. 3B comprises a series of photoeffect contour plots of cellresponse to QDs as a function of QD concentration and light exposureduration. CdTe-2.4 and CdSe-2.4 were plotted in units of toxicity, whileCIS-1.9 was plotted in units of proliferation. Grey color indicates aresponse was within the error threshold of the measurement (p>0.05).FIG. 3C comprises a series of images of cell colonies that were platedand grown following exposure to quantum dots and light.

FIG. 4 comprises a series of photoeffect contours comparing the effectof changing quantum dot size for the materials shown in FIGS. 3A-3C.

FIGS. 5A-5B comprise a series of graphs illustrating phototoxicity forCdTe-2.4 particles in MDR clinical strains. C1 was 10 nM, C2 was 50 nM,and C3 was 100 nM. Data were normalized to t=0 and the average of 3biological replicates, and error bars represent standard deviation.Ordinates represent hours (h).

FIGS. 6A-6B comprise a series of graphs illustrating phototoxicity forCdSe-2.4 particles in MDR clinical strains. C1 was 10 nM, C2 was 50 nM,and C3 was 100 nM. Data were normalized to t=0 and the average of 3biological replicates, and error bars represent standard deviation.Ordinates represent hours (h).

FIGS. 7A-7B comprise a series of graphs illustrating photoproliferationfor CIS-1.9 particles in MDR clinical strains. C1 was 10 nM, C2 was 50nM, and C3 was 100 nM. Data were normalized to t=0 and the average of 3biological replicates, and error bars represent standard deviation.Ordinates represent hours (h).

FIGS. 8A-8B comprise a series of graphs illustrating phototoxicity forAg₂S-1.7 particles in MDR clinical strains. Concentrations wererepresented by dilution from stock of particles where C1 was 1:100dilution, C2 was 1:1,000 dilution, and C3 was 1:10,000. Data were theaverage of 3 biological replicates, and error bars represent standarddeviation. Ordinates represent hours (h).

FIGS. 9A-9B comprise a series of graphs illustrating phototoxicity forCu₂S-2.1 particles in MDR clinical strains. Concentrations wererepresented by dilution from stock of particles where C1 was 1:100dilution, C2 was 1:1,000 dilution, and C3 was 1:10,000. Data werenormalized to t=0 and the average of 3 biological replicates, and errorbars represent standard deviation. Ordinates represent hours (h).

FIGS. 10A-10B comprise a series of graphs illustrating phototoxicity forFeS-2.0 particles in MDR clinical strains. Concentrations wererepresented by dilution from stock of particles where C1 was 1:100dilution, C2 was 1:1,000 dilution, and C3 was 1:10,000. Data werenormalized to t=0 and the average of 3 biological replicates, and errorbars represent standard deviation. Ordinates represent hours (h).

FIG. 11 comprises a series of fluorescence images of HEK293 cells and E.coli grown in co-culture. HEK293 cells were tagged with DAPI (blue) andPhalloidin Cruzfluor 488 conjugate (green), and E. coli were engineeredto express mCherry (red). Scale bars were 200 μm in each image.

FIG. 12 comprises an absorbance spectra and STS measured redoxpotentials of silver, iron, and copper sulfides with comparison to thematerials discussed elsewhere herein. Redox levels associated withphotoproliferation (dashed) and phototoxicity (solid) were marked.

FIG. 13 comprises STM images of (a) 2.6 eV CdSe, (b) 2.4 eV CdSe, (c)2.4 eV CdTe, (d) 2.2 eV CdTe, (e) 1.6 eV CuInS₂. Scale bars were 50 nmin each image. Individual QDs were circled in blue.

FIG. 14 comprises a graph illustrating a lamp emission spectrum (red, A)and filter absorbance spectra (IR—black, B, UV—blue, C).

FIGS. 15A-15B comprise a series of graphs illustrating size distributionhistograms of various quantum dots.

FIGS. 16A-16C comprise graphs illustrating individual replicates of CISphotoresponse. FIG. 16D comprises a graph illustrating proliferationwith statistically significant points (p<0.05) marked.

FIG. 17 illustrates plates of culture shown in FIG. 3C under darkconditions. (a) No treatment, (b) 75 nM CdTe, (c) 125 nM CdSe, and (d)100 nM CuInS₂.

FIG. 18 illustrates images of HEK293 cells after exposure to quantumdots.

FIGS. 19A-19B comprise graphs illustrating changes in CdSe absorbanceover time in light and dark while in the 37° C. incubator.

FIGS. 20A-20B comprise graphs illustrating absorbance spectra of theCdTe quantum dots over time light and dark while in the 37° C.incubator. On the bottom of FIGS. 20A-20B are plots tracking the peakposition of the PL emission of the two sizes over time.

FIG. 21 comprises a graph illustrating absorbance spectra after 24 hoursof incubation of the 1.9 eV CuInS₂ particles.

FIG. 22 comprises graphs illustrating degradation profiles of the CdTecores in light and dark conditions.

FIG. 23A comprises graphs illustrating optical properties of CdTe-2.4coated with MPA and cysteamine (CA). FIG. 23B comprises graphsillustrating degradation profiles of the CA-coated CdTe particles inlight and ark. FIG. 23C comprises a graph illustrating optical densitygrowth curves of MG1655 E. coli exposed to the CA-coated QDs in lightand dark. FIG. 23D comprises a graph illustrating inhibition as afunction of quantum dot concentration. FIG. 23E comprises a graphillustrating uptake of the MPA (−) and CA (+) coated QDs.

FIG. 24A comprises a graph illustrating optical spectra and quantumyields of the CdTe cores and ZnS@CdTe core-shells. FIG. 24B comprises adiagram illustrating band alignment of CdTe with ZnS and the interfacematerials. FIG. 24C comprises a graph illustrating degradation profilesof the core-shells in light and dark.

FIG. 25A comprises a graph illustrating optical density growth curves ofMG1655 E. coli exposed to the core-shells in light and dark. FIG. 25Bcomprises a graph illustrating inhibition as a function of quantum dotconcentration. FIG. 25C comprises a graph illustrating uptake of thecore-shells compared to cores.

FIG. 26A comprises a set of graphs illustrating optical properties andfluorescence quantum yield of the Cd@CdTe particles compared to cores.FIG. 26B comprises a set of graphs graph illustrating emission peakchanges during exposure to PBS in light and dark conditions of thecore-shells. FIG. 26C comprises a graph illustrating optical densitygrowth curves of MG1655 E. coli exposed to Cd@CdTe. FIG. 26D comprises agraph illustrating inhibition as a function of quantum dotconcentration. FIG. 26E comprises a graph illustrating uptake of thecore-shells compared to cores.

FIG. 27A comprises a schematic representation of the finding thatcombination of nanoparticles with antibiotics provides an efficaciouscombination therapy that works to inhibit multi-drug resistant bacteria.FIG. 27B comprises a series of graphs that illustrate characterizationof multidrugs resistant strains used in the study, showing the highlevel of resistance to many classes of antibiotics. Five antibiotics areexemplified. FIGS. 27C-27D comprise a set of graphs illustrating thefinding that combination of streptomycin with CdTe-2.4 quantum dot atvaried antibiotic and CdTe-2.4 concentrations has significant inhibitoryantibacterial effect.

FIG. 28A comprises a set of images illustrating the combination ofCdTe-2.4 with respective antibiotic (shown as the IC₅₀ of the straindivided by the CLSI or breakpoint) and respective strain represented asthe OD at 8 h with treatment divided by the OD at 8 h in no treatment. Acolor of white or red indicates the combinations effect is at or greaterthan the IC₅₀ of the combination. In certain cases the combinationlowers the IC₅₀ of the strain to below the CLSI or breakpoint value, asnoted by they axis of the heat maps which is IC₅₀ of the strain for theantibiotic divided by the CLSI or breakpoint value seen in FIG. 27B.FIG. 28B comprises a set of graph illustrating colony forming unitanalysis for 3 combinations in the respective strain, showingsignificant cell death with combination compared to monotherapy

FIG. 29 comprises a set of images illustrating multi-drug resistantstrains treated with CdTe-2.4 for 2 hours and stained with DCFH-DA. Theresults suggest that the mechanism of action comprises production ofreactive oxidative species upon illumination inside the cell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to the fact thattherapeutics that alter redox homeostasis can be used to target uniquecellular redox environments. In certain embodiments, the tunability ofredox potentials of quantum dots (QDs) can be used to specificallyperturb redox environments that are unique to cell types.

QDs are nanoparticles that are tunable due to quantum confinementeffects resulting from their small size. Their dimensions allow them todiffuse across membranes and accumulate in the intracellular environmentor associate with cellular outer membranes. Further, the tunability ofQDs allows for the control of their optical absorption and band edgeredox potentials. For example, as illustrated in FIG. 1A, three distinctQD materials have shifted band edge redox potentials, even though theyhave the same optical bandgap. This redox tunability allows fortargeting specific cellular processes upon light stimulation and theinducement of desired effects.

As described herein, QDs induce light-activated reactive species (LARS)within cells, and have distinct effects on the cell, depending on thecell type studied. In certain embodiments, LARS generated from tuned QDsare herein alter cellular phenotype; QDs upon illumination with visiblelight may be phototoxic, benign, or photo-proliferative to Escherichiacoli cells (FIG. 1B). As demonstrated herein, this cellular phenotypetuning is not a material-specific property but is indeed dependent onthe tuned electronic properties of the QDs. Further, the resultspresented herein show selective, redox tuned cell death with co-culturestudies of bacteria and mammalian, indicating that the compositions ofthe invention can be used therapeutically.

Further, as described herein, the compositions of the invention can becombined with one or more antibiotic agents, and such combinations areeffective in killing, or preventing and/or hampering the growth ofgram-negative bacteria. In certain embodiments, the gram-negativebacteria are multi-drug resistant. In other embodiments, theconcentration or amount of the antibacterial agent in the combinationsof the invention that kill, or prevent and/or hamper the growth ofgram-negative bacteria, is lower than the concentration or amount of theantibacterial agent that is required to kill, or prevent and/or hamperthe growth of gram-negative bacteria when the antibacterial agent isused alone.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

As used herein, unless defined otherwise, all technical and scientificterms generally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in biology,chemistry and material science are those well-known and commonlyemployed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e. to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein, “about” when referring to a measurablevalue such as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more specifically ±5%, even morespecifically ±1%, and still more specifically ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, the term “band edge redox potential” for a materialcorresponds to the band edge of the conduction band state (reductionpotential) and the band edge of the valence band state (oxidationpotential).

As used herein, the term “electromagnetic radiation” includes radiationof one or more frequencies encompassed within the electromagneticspectrum. Non-limiting examples of electromagnetic radiation comprisegamma radiation, X-ray radiation, UV radiation, visible radiation,infrared radiation, microwave radiation, radio waves, and electron beam(e-beam) radiation. In one aspect, electromagnetic radiation comprisesultraviolet radiation (wavelength from about 10 nm to about 400 nm),visible radiation (wavelength from about 400 nm to about 750 nm) orinfrared radiation (radiation wavelength from about 750 nm to about300,000 nm). Ultraviolet or UV light as described herein includes UVAlight, which generally has wavelengths between about 320 and about 400nm, UVB light, which generally has wavelengths between about 290 nm andabout 320 nm, and UVC light, which generally has wavelengths betweenabout 200 nm and about 290 nm. UV light may include UVA, UVB, or UVClight alone or in combination with other type of UV light. In oneembodiment, the UV light source emits light between about 350 nm andabout 400 nm. In some embodiments, the UV light source emits lightbetween about 400 nm and about 500 nm.

As used herein, the term “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionthat may be used to communicate the usefulness of the compositionsand/or methods of the invention. In certain embodiments, theinstructional material may be part of a kit useful for generatingcompositions of the invention. The instructional material of the kitmay, for example, be affixed to a container that contains thecompositions of the invention or be shipped together with a containerthat contains the compositions. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the recipient uses the instructional material and the compositionscooperatively. For example, the instructional material is for use of akit; instructions for use of the compositions; or instructions for useof the compositions.

As used herein, the term “LARS” refers to light-activated reactivespecies.

As used herein, the term “MPA” refers to 3-mercaptopropionic acid.

As used herein, the term “QD” refers to quantum dot.

Throughout this disclosure, various aspects of the invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range and, when appropriate,partial integers of the numerical values within ranges. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and so on, as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

DISCLOSURE

The present invention comprises, in one aspect, a novelnanoparticle-based therapeutic strategy using light-activated quantumdots (QDs) that specifically tune phenotypic responses of Escherichiacoli and HEK293 to changes in redox properties of nanomaterials. Theresulting light-activated reactive species are selectively phototoxic,benign, or photo-proliferative depending on the quantum dot redoxpotentials and cell type. The photoeffects observed in cellularphenotype are not material-dependent properties, but rather due to thetuned electronic properties of the QDs. The present studies demonstratethat the compositions of the present invention can be used in apotential therapeutic intervention, and show selective, redox tuned celldeath with co-culture studies of E. coli and HEK293 cells. In certainembodiments, the compositions of the invention can be used asantimicrobial agents that selectively targets the redox environment ofbacteria and not that of mammalian cells. In other embodiments, thecompositions of the invention can be used to alter the redox state ofany potential organism of interest. Changes in redox homeostasis ofhuman cells can cause enhanced cell proliferation by interfering insignal transduction pathways in the development of cancer or by creatinga reducing environment. This use has application throughout thetherapeutic field, as well as a tool for studying the effect of redoxstates on cellular phenotypes.

As demonstrated herein, the dependence of cellular effect on the quantumdot oxidation and reduction potentials was confirmed, decoupling theeffect from the material and the bandgap. A novel photo-proliferativeeffect with CIS-1.9 particles was observed, where cell growth wasenhanced upon light stimulation and the resulting LARS. The tunedCIS-1.9 particle is the first QD study to demonstrate this effect. Takenas a whole, the present results indicate that the compositions andmethods of the invention can be used for selective phenotypic tuning ofcells.

Compositions

The invention includes a semiconductor-containing nanoparticle, whereinthe at least one nanoparticle has a band edge redox potential such thatirradiation of the composition with radiation ranging from about 400 nmto about 1,000 nm in the presence of a first cell, under conditionswhereby the at least one nanoparticle penetrates the first cell,promotes growth, or kills and/or prevents growth, of the first cell. Theinvention further includes any compositions including suchnanoparticles.

In certain embodiments, the at least one nanoparticle comprises aquantum dot (QD). In other embodiments, the composition is irradiatedwith radiation ranging from about 750 nm to about 1,000 nm. In otherembodiments, the composition further comprises the first cell. In yetother embodiments, the first cell is a bacterium. In yet otherembodiments, the bacterium comprises at least one selected from thegroup consisting of K. pneumonia, E. coli, S. aureus, P. aeruginosa, A.baumannii and S. typhimurium.

In certain embodiments, the composition further comprises a second cell,wherein irradiation of the composition has no measurable effect on thegrowth, metabolism and/or survival of the second cell. In otherembodiments, the second cell is mammalian.

In certain embodiments, irradiation of the composition in the presenceof the first cell promotes growth of the first cell, and the QDcomprises CuInS₂. In other embodiments, irradiation of the compositionin the presence of the first cell kills and/or prevents growth of thefirst cell, and the QD comprises CdTe. In yet other embodiments,irradiation of the composition in the presence of the first cellpromotes growth of the first cell, and the band edge of the conductionband state (reduction potential) of the QD is about +0.2 V and the bandedge of the valence band state (oxidation potential) of the QD is about−1.8 V, as referenced to NHE (standard hydrogen electrode). In yet otherembodiments, irradiation of the composition in the presence of the firstcell promotes death and/or prevent growth of the first cell, and theband edge of the conduction band state (reduction potential) of the QDis about +0.35 V and the band edge of the valence band state (oxidationpotential) of the QD is about −2.1 V, as referenced to NHE (standardhydrogen electrode).

In certain embodiments, irradiation of the composition changes redoxhomeostasis in the first cell. In other embodiments, irradiation of thecomposition generates at least one light-activated reactive species inthe first cell.

In certain embodiments, the surface of the at least one negativelycharged nanoparticle is overall negatively charged. In otherembodiments, the surface of the at least one negatively chargednanoparticle is essentially free of positively charged ligands. In yetother embodiments, the surface of the at least one negatively chargednanoparticle is free of positively charged ligands.

In certain embodiments, the at least one nanoparticle comprises CdTe,and the surface of the at least one nanoparticle is at least partiallycoated with ZnS. In other embodiments, the at least one nanoparticlecomprises CdTe, and the surface of the at least one nanoparticle isessentially fully coated with ZnS. In yet other embodiments, the atleast one nanoparticle comprises CdTe, and the surface of the at leastone nanoparticle is fully coated with ZnS. In other embodiments, thefluorescent quantum yield of the at least one nanoparticle at leastpartially coated with ZnS is similar, or essentially identical, to thatof the at least one nanoparticle without the ZnS coating.

In certain embodiments, the at least one nanoparticle comprises CdTe,and the surface of the at least one nanoparticle is at least partiallycoated with CdS. In other embodiments, the at least one nanoparticlecomprises CdTe, and the surface of the at least one nanoparticle isessentially fully coated with CdS. In yet other embodiments, the atleast one nanoparticle comprises CdTe, and the surface of the at leastone nanoparticle is fully coated with CdS. In other embodiments, thefluorescent quantum yield of the at least one nanoparticle at leastpartially coated with CdS is similar, or essentially identical, to thatof the at least one nanoparticle without the CdS coating.

In certain embodiments, the nanoparticle of the invention is combinedwith one or more antibacterial agents, and such combinations kill, orprevent and/or hamper the growth of gram-negative bacteria whenirradiated. In other embodiments, the gram-negative bacteria aremulti-drug resistant. In yet other embodiments, the concentration oramount of the antibacterial agent in the combinations of the inventionis lower than the concentration or amount of the antibacterial agentthat is required to kill, or prevent and/or hamper the growth ofgram-negative bacteria when the antibacterial agent is used alone.

In certain embodiments, the antibacterial agent comprises acephalosporin antibiotic, fluoroquinone, or protein synthesis inhibitor.In other embodiments, non-limiting examples of antibacterial agentsuseful for treating gram-negative bacterial infections include:Amikacin, Aztreonam, Cefdinir, Cefaclor, Cefamandole, Cefditoren,Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil,Cefuroxime, Ceftazidime, Ceftibuten, Ceftobiprole, Ceftriaxone,Chloramphenicol, Ciprofloxacin, Clindamycin, Colistin, Ertapenem,Doripenem, Gatifloxacin, Gentamicin, Imipenem/Cilastatin, Kanamycin,Levofloxacin, Meropenem, Metronidazole, Moxifloxacin, Neomycin,Netilmicin, Ofloxacin, Paromomycin, Polymyxin B, Streptomycin,Thiamphenicol, Tigecycline, and Tobramycin.

Methods

The invention includes a method of promoting growth, or killing orpreventing growth, of a first cell. The invention further includes amethod of altering redox homeostasis in a first cell.

In certain embodiments, the method comprises irradiating the first cellwith radiation ranging from about 400 nm to about 1,000 nm in thepresence of at least one semiconductor-containing nanoparticle with aband edge redox potential, under conditions whereby the at least onenanoparticle penetrates the cell.

In certain embodiments, the method comprises irradiating the first cellwith radiation ranging from about 400 nm to about 1,000 nm in thepresence of at least one semiconductor-containing nanoparticle with aband edge redox potential, under conditions whereby the at least onenanoparticle penetrates the first cell, whereby growth of the first cellis promoted, growth of the first cell is prevented or killing of thefirst cell is promoted.

In other embodiments, the method comprises irradiating the first cellwith radiation ranging from about 400 nm to about 1,000 nm in thepresence of at least one semiconductor-containing nanoparticle with aband edge redox potential, under conditions whereby the at least onenanoparticle penetrates the first cell, whereby redox homeostasis in thefirst cell is altered.

In certain embodiments, the nanoparticle comprises a quantum dot (QD).In other embodiments, the first cell is irradiated with radiationranging from about 750 nm to about 1,000 nm. In yet other embodiments,the first cell is a bacterium. In yet other embodiments, the bacteriumcomprises at least one selected from the group consisting of K.pneumonia, E. coli, S. aureus, P. aeruginosa, A. baumannii and S.typhimurium.

In certain embodiments, the first cell is in the presence of a secondcell, and irradiation of the first and second cells in the presence ofthe at least one nanoparticle has no measurable or significant effect onthe growth, metabolism and/or survival of the second cell. In otherembodiments, the second cell is mammalian. In yet other embodiments,growth of the first cell is promoted, and the QD comprises CuInS₂. Inyet other embodiments, growth of the first cell is prevented or killingof the first cell is promoted, and the QD comprises CdTe. In yet otherembodiments, growth of the first cell is promoted, and the band edge ofthe conduction band state (reduction potential) of the QD is about +0.2V and the band edge of the valence band state (oxidation potential) ofthe QD is about −1.8 V, as referenced to NHE (standard hydrogenelectrode). In yet other embodiments, growth of the first cell isprevented or killing of the first cell is promoted, and the band edge ofthe conduction band state (reduction potential) of the QD is about +0.35V and the band edge of the valence band state (oxidation potential) ofthe QD is about −2.1 V, as referenced to NHE (standard hydrogenelectrode).

In certain embodiments, irradiation changes redox homeostasis in thefirst cell. In other embodiments, irradiation generates at least onelight-activated reactive species in the first cell.

In certain embodiments, the first cell is further contacted with atleast one antibacterial agent. In other embodiments, the first cellcomprises a gram-negative bacterium. In yet other embodiments, the firstcell comprises a multi-drug resistant gram-negative bacterium. In yetother embodiments, the concentration or amount of the antibacterialagent that is required to kill, or prevent and/or hamper the growth ofgram-negative bacteria in the presence of the at least one nanoparticleis lower than the concentration or amount of the antibacterial agentthat is required to kill, or prevent and/or hamper the growth ofgram-negative bacteria when the antibacterial agent is used alone.

In certain embodiments, the antibacterial agent comprises acephalosporin antibiotic, fluoroquinone, or protein synthesis inhibitor.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds are intended to be exemplary, as it is known that oneof ordinary skill in the art can name the same compounds differently.When a compound is described herein such that a particular isomer orenantiomer of the compound is not specified, for example, in a formulaor in a chemical name, that description is intended to include eachisomers and enantiomer of the compound described individual or in anycombination. Although the description herein contains many embodiments,these should not be construed as limiting the scope of the invention butas merely providing illustrations of some of the presently preferredembodiments of the invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication. In general the terms and phrases used herein have theirart-recognized meaning, which can be found by reference to standardtexts, journal references and contexts known to those skilled in theart. Any preceding definitions are provided to clarify their specificuse in the context of the invention.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials:

3-Mercaptopropionic acid (>99%; also known as MPA) was purchased fromAcros Organics. Cadmium(II) chloride (technical grade), 10 mMphosphate-buffered saline, oleic acid (90%), copper(II) acetylacetonoate(?99.99%), indium(III) acetate (99.99%), sulfur (99.5%), and oleylamine(technical grade) were purchased from Sigma Aldrich. Tellurium-325 meshpowder (99.99% metal basis), and selenium-325 mesh powder (99.5%) werepurchased from Alfa Aesar. Sodium borohydride (98%), and sodiumhydroxide (>97.0%), were purchased from Fisher Scientific. Compressednitrogen (pre-purified), and oxygen gas (ultra-high purity) werepurchased from Airgas. Ethanol (200 proof) was purchased from DeconLaboratories INC. All purchased materials were used as provided withoutfurther purification.

CdTe and CdSe Quantum Dot Synthesis and Sterilization:

For CdTe, deionized water was initially degassed using bubbling nitrogenfor 30 min. 1 mL degassed water was used to dissolve NaBH₄ (35 mg, 0.93mmol), and the resulting solution was transferred to a septum-capped 2mL vial (Thermo Scientific) containing Te powder (40 mg, 0.31 mmol). Te(325 mesh) was required for the reaction, as coarser Te would not reactwell.

A needle was inserted into the septum for outgassing during thereaction, which was allowed to proceed until the tellurium precursorsolution became optically clear and colorless (40-60 min). A cadmiumprecursor solution was created by dissolving CdCl₂ (3.7 mg, 0.020 mmol)and 3-mercaptopropionic acid (MPA, 1.8 μL, 2.2 mg, 0.021 mmol) in 10 mLof degassed water. The reaction solution was made by mixing 250 μL ofthe cadmium precursor solution, 250 μL degassed water, 1 μL of thetellurium precursor solution, and 10 μL of 0.5 M NaOH (total volume 511μL). Reactions were scaled up to a maximum of 1.5 mL total volume. 100μL aliquots of the reaction solutions were divided into PCR tubes(Thermo Scientific), and placed in a thermocycler (Bio-Rad T100). Thetubes were held at 98° C. for the reaction duration (1 hour for greenemitting CdTe, 2 hours for yellow, 5 hours for red). The quantum dotswere then filtered (Omega 4K Nanosep), and washed with pH 10 water. Thepurified dots were re-dispersed in 150 μL pH 10 water for storage. CdSewas prepared using the same procedure using various masses of Se (25 mg,0.32 mmol) and NaBH₄ (25 mg, 0.66 mmol), the reaction between the twooccurring at a much higher rate (<10 min). General procedure was adaptedfrom Tikhomirov, et al., 2011, Nature Nanotech. 6:485-490.

Prior to integration with cells, the CdX quantum dots were sterilized inthe following manner. 300 μL of stock QD solution in pH 10 aqueous mediawere filtered to dryness in a 4K filter. The dots were then washed with200 μL of ethanol and filtered to dryness. A final wash with 200 μL ofautoclaved PBS solutions completed the process, and the dots werere-suspended in PBS. The concentrations of these purified solutions weredetermined optically using published correlations (Yu, et al., 2003,Chem. Mater. 15:2854-2860).

CuInS₂ Quantum Dot Synthesis and Ligand Exchange:

A 100 mL three-necked flask was charged with copper(II) acetylacetonoate(260 mg, 1.0 mmol), indium(III) acetate (290 mg, 1.0 mmol), oleylamine(1.0 mL, 1.2 g, 4.5 mmol), and o-dichlorobenzene (7 mL). The flask wasthen connected to a Schlenk line and purged with alternating vacuum andnitrogen refilling. After three cycles the temperature was increased to110° C. using a J KEM Scientific Model 210 temperature controller. Thesulfur precursor solution was made by dissolving sulfur (64 mg, 2 mmol)in o-dichloro benzene (3 mL) via gently heating. Once dissolved, thesulfur was rapidly injected into the reaction flask and the temperaturewas increased to 180° C. for the duration of CIS growth. Once thedesired reaction time had elapsed the flask was quenched in a waterbath, and the contents transferred to a centrifuge tube. Excess ethanolwas added and the mixture was centrifuged at 5,000 RPM for 5 min. Theprecipitated particles were then re-dispersed in chloroform, andcentrifuged again to remove poorly passivated dots. Dots that remainedin solution were stored for further use along with excess oleylamine topromote stability. Procedure adapted from Panthani, et al., 2008, J. Am.Chem. Soc. 130:16770-16777.

The long-chain amine ligands were exchanged with MPA in the followingmanner. The hexane stock solution (100 μL), 0.5M NaOH (200 μL), ethanol(500 μL), and MPA (400 μL) were mixed in a 1.5 mL Eppendorf tube thatwas placed on an Eppendorf Mixmate at 1,000 RPM for 3 hours. The tubeswere then centrifuged at 10,000 RPM for 15 min at 10° C. The liquidphase was then removed completely, and the precipitated dots wereconcentrated in a small volume (<50 μL) of ethanol. This was transferredto a new sterile tube and was vacuum dried to yield a powder. SterilePBS was then used to re-disperse the dots for use with cells.Concentrations were determined optically using published correlations(Booth, et al., 2012, Chem. Mater. 24:2064-2070). This procedure yieldssterile dots, and was scaled by adding more reaction tubes.

Metal Sulfide Syntheses:

Deionized water was initially degassed using bubbling nitrogen for 30min. A second aliquot of 10 mL deionized water was then degassed in thesame way. In this 10 mL metal salts were dissolved (AgNO₃: 5.5 mg,FeCl₂: 4.0 mg, CuCl was added until saturated) to form the metalprecursor solution. 30 μL, 0.5 M NaOH and 750 μL, DI water were added toa 1.5 mL Eppendorf tube and placed in an ice bath. 2 μL,3-mercaptopropionic acid were added to the metal precursor solution, and750 μL, of which were added to the Eppendorf tube. 1.5 μl of 630 mMammonium sulfide solution was then added and the vessel mixed. The tubewas left in the ice bath for 30 min before being removed for furtherexperiments, the same sterilization method was employed as used forCdSe/CdTe.

Core Synthesis:

CdTe and CdSe cores of various sizes were synthesized using thematerials and methods described elsewhere herein.

Cysteamine Ligand Exchange:

A stock of cysteamine-hydrochloride (CA) was created by dissolving CA(7.7 mg, 0.10 mmol) in 1 mL of 0.1 M HCl, and the pH was adjusted to 6.This was used to re-disperse CdTe-2.4 cores which were filtered, andwashed twice with double-distilled water (ddH₂O). The QDs were then keptin the dark at room temperature overnight. Prior to use the particleswere bulk-centrifuged at 10 krpm for 5 min to remove poorly-passivatedQDs, and washed in a similar manner using PBS.

ZnS Core-Shell Synthesis:

A stock 100× solution of zinc and sulphur sources was created bydissolving Zn(NO₃)₂.6H₂O (609 mg, 5.57 mmol) and thiourea (75 mg, 1.0mmol) in 10 mL ddH₂O. For a synthesis, 100 μL of the 100× stock wasdiluted into 10 mL of freshly de-gassed ddH₂O which served as thezinc-sulphur precursor stock. 200 μL of CdTe-2.4 stock were filtered,washed twice and re-dispersed with pH 11 water. This solution was thendiluted to 2 μM. The reaction solution consisted of the filtered quantumdots and the precursor stock in a 1:1 ratio, with 10 μL of 0.5 M NaOHper 500 μL of reaction volume. This mixture was then divided into 100 μLPCR tubes and reacted at 98° C. for 1 h. Prior to use in cell culturesthey were filtered and washed as described elsewhere herein.

Cd-Overcoat Synthesis:

A Cd-MPA stock was prepared and degassed as described elsewhere hereinfor the core syntheses. 200 μL of CdTe-2.4 stock were filtered, washedtwice, re-dispersed with pH 11 water, then diluted to 2 μM. The QD andCd-MPA stocks were mixed in equal volumes with 10 μL of 0.5 M NaOH per500 μL of reaction volume. The reaction solution was then divided into100 μL PCR tubes and reacted at 98° C. for 15 min.

Scanning Tunneling Spectroscopy:

All STM/STS measurements were taken with a modified Molecular ImagingPicoSPM II Microscope and controller. STM images were taken using aPt—Ir tip (80:20, Agilent Technologies) with a sample bias of +1.0V anda set-point tunneling current of +0.5 nA (FIG. 13). Indium-tin-oxide(ITO) substrates (Delta Technologies) were prepared prior to use bywashing with ethanol, then cleaning by O₃ plasma for 5 min (JelightCompany INC UVO Cleaner Model No. 42). Each QD sample was drop cast ontothe ITO (10-20 μL of 100-500 nM solution). Scanning tunneling spectrawere acquired by varying the voltage across the sample and Pt—Ir tip.The DOS was calculated from the first derivative of the current-voltagecurves. The error bars in FIG. 2C represent the spread in CB/VBpositions over ˜20 independent quantum dot measurements.

Light Source for Cell Studies:

Cells were illuminated using a tungsten lamp (GE 35200-EKE) placedexternally of the incubator via a fiber optic cable. The lamp wasequipped with filters to remove UV (Thorlabs FEL0400) and IR light,creating a bandpass filter from 400-700 nm (FIG. 14). The lamp wasoperated at 75% of maximum power for all experiments.

Transmission Electron Microscopy:

TEM images of the nanoparticles were obtained on a Philips CM 100, andwere used for confirmation of QD shape and size. CdSe and CIS particleswere imaged at 60 kV while CdTe was imaged at 80 kV. Particle sizedistributions were determined using ImageJ (FIGS. 15A-15B).

Absorbance and Photoluminescence Measurements:

UV-VIS spectra were acquired on a VWR UV1600-PC spectrophotometer at 1nm resolution. Photoluminescence spectra were measured by illuminatingthe sample with a UV lamp (UVP UVGL-25) and collecting the resultingemission spectrum with an Ocean Optics USB 4000 detector.

Quantum Yields (QY) were Determined Via Comparison with a Fluoresceinisothiocyanate (FITC, Sigma) standard using a NIST calibrated PhotonTechnologies International fluorimeter for each sample, with emissionmeasured from 485-800 nm using 475 nm excitation. Each QY was calculatedusing Equation (1). The emission spectra used in the degradation studieswere obtained using a calibrated Ocean Optics USB4000 detector.

$\begin{matrix}{\frac{\Phi_{QD}}{\Phi_{FITC}} = \frac{A_{FITC}{\int_{485}^{800}{I_{QD}\lambda \; d\; \lambda}}}{A_{QD}{\int_{485}^{800}{I_{FITC}\lambda \; d\; \lambda}}}} & (1)\end{matrix}$

Bacterial Strains and Cell Culture Conditions:

Zymo DH5-α E. coli cells were used, and individual colonies wereselected for each replicate. Liquid cultures were grown overnight in 2%lysogeny broth (LB) (incubated at 37° C.), diluted 1:10 into LB withrespective quantum dots, and rocked. Solid cultures were grown on 2% LBbroth, 1.5% agar at 37° C. Optical density measurements were taken atusing a Tecan GENios 562 nm with a bandwidth of 35 nm. All bacterialfreezer stocks were stored in 40% glycerol at −80° C.

All multi-drug-resistant (MDR) clinical strains were obtained as a giftfrom Dr. Nancy Madginer at the University of Colorado, Denver. MDRstrains were cultured in cation adjusted Mueller Hinton broth (CAMHB)liquid or 1.5% agar solid for all studies. Cultures were started fromindividual colonies and grown overnight in 1 mL CAMHB. Bacteria wasdiluted 1:10 from the overnight for photoeffect experiments. Photoeffectexperiments were carried out in 50 uL cultures in 384 well transparentflat bottom plates. Optical density measurements were taken using aTecan GENios at 562 nm with a bandwidth of 35 nm. All MDR bacterialfreezer stocks were stored in 10% glycerol at −80° C.

TABLE 1 MDR clinical isolates Graph label Strain Special CharacteristicsS1 MDR Klebsiella pneumoniae NDM-1 S2 MDR E. coli Carbapenem resistantS3 MDR Enterococcus faecalis Vancomycin resistant S4 MDR Staphylococcusaureus Methicillin resistant (MRSA) S5 MDR K. pneumoniaeExtended-spectrum β-lactamases S6 MDR Pseudomonas aeruginosa S7 MDR K.pneumoniae S10 MDR Salmonella typhimurium Extended-spectrum β-lactamasesS12 MDR E. coli S13 MDR Acinetobacter baumannii complex

At least for Examples 7-9, colonies of E. coli (MG1655) were grown onsolid Luria Bertani (LB, Sigma Aldrich)-agar media overnight at 37° C.from freezer stocks (40% glycerol, −80° C.) and stored at 4° C. For amicroplate assay, three individual colonies were grown overnight in LB,and diluted 1:100 when incorporated with the various quantum dots.Separate 96-well flat-bottom plates were prepared for light and darkconditions, the OD of which were measured using a Tecan GENios at 562nm. Plates were shaken at 225 rpm in a 37° C. incubator betweenmeasurements. The dark plate was wrapped in aluminum foil while the edgeof the light plate was sealed with parafilm to reduce evaporation. Thelight source was modulated before each experiment to provide the desiredintensity, and was equipped with a 400 nm longpass filter (ThorLabsFGL400) and a 300-700 nm bandpass filter (FGS900-A) to remove UV and IRlight.

Statistical Significance of Photoproliferation:

Photoproliferation was analyzed by comparing the average proliferationof the three biological replicates exposed to CuInS₂ (FIGS. 16A-16C) tothe cell colonies that had no treatment. Comparing the proliferation ofthese two groups (FIG. 16D) using two-way ANOVA reveals the time pointsat which statistically significant proliferation occurred.

Colony Forming Unit (CFU) Analysis:

Cultures were sampled at respective time points during a bacterialtoxicity study and serial dilutions were performed ranging from10²-10¹⁰. Dilutions were plated on 2% LB, 1.5% agar, grown at 37° C. for24 hr, and counted (FIG. 17). Images shown in main text are diluted 10³before plating 10 μL.

Mammalian Cell Culture:

HEK-293T cells were used between passages 11-20. Cells were recoveredfrom freezer stocks in high glucose Dulbecco's Modified Eagle Mediumsupplemented with glutamine and fetal bovine serum (FBS). Cultures weregrown at 37° C. in 5% CO₂ with controlled humidity. Cells were passagedat 80% confluency with 0.25% trypsin and seeding densities werecalculated using a hemocytometer. Cells were stored in liquid nitrogenfor long term storage and −80° C. for short term.

Cells were seeded at 6,000 cells per well into a tissue culture treated96-well plate (Cellstar). Media was supplemented with penicillinstreptomycin solution to minimize the chance of contamination. Quantumdot dilutions were made in sterile Dulbecco's modified phosphatebuffered saline (dPBS). Images of these cells were acquired on amicroscope after 24 hours of treatment. Three replicate images weretaken by randomly imaging different locations in each well.Representative images under all QD conditions shown in FIG. 18.

Co-Culture Experiment:

Co-culture experiments were carried out with HEK-293T cells betweenpassage number 18 and 24 and DH5-α E. coli transformed with pFPV-mCherryplasmid (Addgene). The pFPV-mCherry plasmid was used in theseexperiments for the constitutive production of fluorescent proteinmCherry for imaging purposes. 9,000 HEK 293T cells were seeded into 96well plates and allowed to grow for 36 hours. The 96 well plates werepretreated with 0.01% poly-L-lysine for one hour and rinsed twice withdPBS prior to seeding. Separate 96 well plates were used for the lightand dark conditions. pFPV-mCherry E. coli were grown for 16 hr fromcolony under above described bacterial cell culture conditions and with100 μg/mL ampicillin sodium salt to maintain the plasmid. DMEM wasremoved from the HEK 293T cultures and supplemented with DMEM containingand approximately 10⁵ bacterial cells/mL, 100 μg/mL ampicillin sodiumsalt, and respective quantum dots in dPBS. Plates were then placed in anincubator with 5% CO₂ at 37° C. for 24 hr either illuminated or shieldedfrom light with tin foil. Media and/or bacterial culture were removedfrom the wells, pelleted at 7,000 rpm for 5 min, and re-suspended in thesame volume of dPBS.

Mammalian cells were then staining with the following procedure; rinsedwell twice with dPBS and fixed in 4% methanol free formaldehyde for 5min. Rinsed again twice with dPBS and treated with 0.1% triton x-100. Anadditional two rinses with dPBS were followed by staining with a 1×dilution of Phalloidin CruzFluor 488 Conjugate (Santa CruzBiotechnology) for 20 min. After washing twice with dPBS they weretreated with 300 nM DAPI for 5 min. A final set of two washes with dPBSand covering with tin foil to protect stains completes the procedure.

Degradation Studies:

QDs were centrifuged and filtered in the same manner used to preparestocks for biological assays. Two samples of each type were prepared inPBS to simulate a biologically relevant medium. One was kept in dark,while the other was illuminated using the 100% light intensity used inthe assays. Emission spectra were recorded using 365 nm excitation and acalibrated Ocean Optics USB4000 detector.

Uptake Studies:

Three cultures were grown overnight and diluted 1:10 intophosphate-buffered saline with the quantum dots at 100 nM totalconcentration. The cultures were then shaken for 1 h at 37° C. andcollected into centrifuge tubes. The tubes were spun at 15 krpm at 3 minand the supernatant was removed. The cell pellet was then washed twicewith PBS and once with double-distilled water using this procedure. Thepellet was then dispersed in ˜300 μL double-distilled water for storage(final volume was recorded after dispersion).

ICP-MS samples were prepared by diluting 25 μL of the samples to 1 mLtotal volume. Standards were prepared within the limits of the possibleconcentration range for comparison. This analysis provided the rawelement composition of the samples, which was used to calculate thesignal corresponding to specific concentrations. The percentage uptakereported herein are defined using a mass balance comparing the totalnumber of particles associated with the cells with the initial numberintroduced into the cultures.

Quantum Dot Controls:

Without wishing to be limited by any theory, CdX toxicity could inprinciple be attributed to release of free cadmium into theintracellular medium. The rate of cell death does not correlate with theconcentration of free Cd²⁺ (Cho, et al., 2007, Langmuir 23:1974-1980),which indicates this is not the source of toxicity in this concentrationrange. Control measurements were performed to track the changes in theQDs for the duration of the cell exposure. The changes in the quantumdots as a result of continued light illumination were examined byabsorption and photoluminescense measurements. Absorbance measurementsof CdSe particles indicated that the smallest particles were relativelystable in dark and under illuminated reaction conditions, experiencingan attenuation of the excitionic peak slowly over 24 hours ofillumination (FIGS. 19A-19B). The largest CdSe particles were, however,less stable than their smaller counterparts, and experienced significantabsorbance decreases within 5-6 hours of illumination.

While informative that changes to the particles are taking place, theexact nature of those changes were not clear, based on these absorbancemeasurements. Because CdTe is photoluminescent in aqueous media, changesin the PL peak position were tracked over time under the sameconditions. There was an initial red shift of the emitted light, whichwas indicative of defect states forming on the quantum dot surfaces,likely oxygen replacing tellurium (FIGS. 20A-20B). Later, the shiftreversed, such that the emitted light decreases in wavelength. Thisresult is consistent with the continuing oxidation of the quantum dotleading to a smaller CdTe core that emits lower wavelength light. Thisblue shift occurs more rapidly in the larger particles, likely due tothe lower relative passivation of tellurium rich facets. As CdO has verylow solubility in buffered solution, the source of the quantum dottoxicity in light was indeed due to the formation of LARS, and not therelease of free Cd²⁺ ions. Without wishing to be limited by any theory,there was a difference between light and dark exposed quantum dotsinsofar as the intensity of light emission decreased much more rapidlywhen exposed to light; this indicated that the LARS contributed to theformation of less ordered particles, facilitating non-radiativerecombination.

There were less overall changes in the CuInS₂ over time compared to thecadmium based dots (FIG. 21), likely due to the greater oxygen stabilityof sulfur as an anion compared to the other chalcogens.

Example 1: Characterization of Tunable Quantum Dot Properties

To examine the range of possible effects, cadmium telluride (CdTe),cadmium selenide (CdSe), and copper indium sulfide (CuInS₂, CIS) QDs ofdifferent sizes, which absorb light across the visible and near-infraredspectra, were prepared (FIG. 2A). Infrared absorbing dots are highlyattractive, as human tissue is generally transparent to light from750-1000 nm allowing in vivo stimulation.

Electronic structures of these materials were quantified using scanningtunneling spectroscopy of individual nanocrystals (FIG. 2B). For clarityof comparison to biochemical systems, these measurements are comparedrelative to the normal hydrogen electrode potential. Based on thesemeasurements, the band edge states of the various materials, which playthe largest role in determining the redox potentials of each quantumdot, were identified. Because the samples were not perfectlymono-disperse, there was a distribution in band position for each size(FIG. 2C). There was a significant size effect with the positions ofeach material's band edge states. For CdSe and CdTe, a change in sizemost notably changed the position of the conduction states (reductionpotential), while leaving the valence states (oxidation potential)relatively constant. In contrast, both potentials are altered withchanging size in CIS QDs.

Example 2: Photoeffect of Quantum Dots on Escherichia coli

To evaluate the photoeffect of tuned QDs in a biological environment,CdTe and CdSe of the same bandgap (2.4 eV) with varying redox potentialswere selected (FIG. 2c ), hereafter referred to as CdTe-2.4 andCdSe-2.4, respectively. 1.9 eV bandgap CIS particles (FIG. 2C, hereafterreferred to as CIS-1.9) were also evaluated, because they have the samereduction potential as the CdSe-2.4 particle and a lower oxidationpotential than CdSe-2.4 and CdTe-2.4 along with a smaller bandgap. Thecomparison of CdSe-2.4 to CdTe-2.4 allowed for a direct observation ofthe effect of the redox potentials as opposed to the bandgap. Thecomparison of CIS-1.9 to CdSe-2.4 allows for direct observation of theeffect of the oxidation potential as the reduction potential is the samefor both particles.

QDs were added, in varying concentrations, to E. coli cultures and grownrich media while the optical density was monitored using a microplatereader. Cultures were grown in the absence and presence of QDs with andwithout visible light illumination (lamp spectra with UV and IR filters,FIG. 14).

Exposure in dark was used as a control, as cadmium based QDs can beintrinsically toxic to cells; however, the present studies wereperformed below reported toxic levels. Further, any intrinsic toxicitywould be accounted for in the dark control analysis. Degradation studiesof cadmium QDs upon light illumination showed a red shift in quantum dotemission as opposed to a blue shift that would be indicative of free,toxic cadmium release (FIGS. 19A, 19B, 20A, 20B, 21A, 21B).

The studies showed a significant reduction in growth of E. coli grown inthe presence of CdTe-2.4 particles in light compared to dark (FIG. 3A).If the phototoxicity were an effect of generic ROS, as opposed to thetuned response, higher phototoxicity with illumination of CdSe-2.4particles would be expected due to their higher oxidation potential. Incontrast to this hypothesis, in the presence of CdSe-2.4 andillumination, no significant phototoxicity was observed in E. coli (FIG.3A). Surprisingly, CIS-1.9 particles upon illumination displayed aphoto-proliferative effect in E. coli.

Evaluating these particles over a concentration range allowed for thedetermination of the threshold where a photoeffect was observed. Inorder to assess the photoeffect of the QDs, parameters for phototoxicity(Equation 2) and photo-proliferation (Equation 3) were defined toobserve the optical density of cultures in light relative to dark. Therewas a strong phototoxic effect above 35 nM CdTe-2.4 (FIG. 3B).Contrastingly, there was no significant photoeffect even up to aCdSe-2.4 concentration of 500 nM (FIG. 3B). Plating the cells postexposure confirmed that the CdTe-2.4 is bactericidal in light and not indark, as evidenced by a large decrease in the number of viable cellswhereas CdSe-2.4 did not exhibit a bactericidal photoeffect (FIGS. 3Cand 17). Thus, the source of the photoeffect is the specifically tunedredox potentials of the QDs and not the bandgap, which remained constantat 2.4 eV.

Phototoxicity=[(OD _(dark) /OD _(light))−1]×100  (2)

Photo-proliferation=[(OD _(light) /OD _(dark))−1]×100  (3)

Examining the concentration dependence of CIS-1.9 showed a statisticallysignificantly region of photo-proliferation centered at 100 nM CIS-1.9(FIG. 3B). There were thresholds below which the generated LARS were attoo low of concentration to greatly impact the cell, and above which theLARS are in excess and overwhelm redox homeostasis. The measuredphoto-proliferation observed with CIS-1.9 differed greatly in the nophotoeffect observed for CdSe-2.4. As the bandgap was not the source ofthe difference in photoeffect between CdTe-2.4 and CdSe-2.4, withoutwishing to be limited by any theory, a possible conclusion is that theCIS-1.9 photo-proliferation compared to CdSe-2.4 derives from the 0.5 Vshift in oxidation potential between the two materials.

Example 3: Confirming E. coli Response is Non-Material Dependent

To observe fine changes in redox potential, various sizes of the threematerials were examined to confirm that these were not materialdependent properties. The focus was on two CdTe particles, CdTe-2.3 witha bandgap of 2.3 eV and CdTe-2.2 with a bandgap of 2.2 eV. A higherenergy CdSe particle with a bandgap of 2.6 eV and a lower energy CISparticle with a bandgap of 1.6 eV, hereafter referred to as CdSe-2.6 andCIS-1.6, respectively, were analyzed as well. CdTe materials showeddecrease in the phototoxic effect with decreasing reduction potential(FIG. 4). As the bandgaps changed with increasing size, their reductionpotentials moved closer to those of CdSe-2.4, implying that in certainembodiments the conduction band states relate to the phototoxic effect,given the consistency in valence band position (FIG. 2C). This datafurther precludes the suggestion that phototoxicity is arising fromdegradation of the QDs, as the larger dots would be more toxic given thegreater amount of material they can release.

Interestingly, CdSe-2.6 retained its benign response, even with itsreduction potential closer to the CdTe-2.4 level, suggesting that incertain embodiments the oxidation potential determined by the valenceband interfered with the generation of toxic LARS. The high selectivityof each redox potential was exemplified by the larger CIS-1.6 QDs. Thesmall change in bandgap led to a slight phototoxic effect, counter towhat was observed for CIS-1.9. Unlike CdTe or CdSe materials, both bandsmoved appreciably with changing size, making neither potential able toactivate a photo-proliferative response.

Example 4: Photoeffect of Quantum Dots on Multi-Drug-Resistant ClinicalStrains of Bacteria

After confirming the non-material dependent qualities of quantum dotsand their photoeffects in a lab strain of E. coli, the particles wereimplemented with multi-drug resistant (MDR) clinical isolates (Table 1).CdTe-2.4 showed significant phototoxic effect for three MDR strains ofK. pneumoniae (S1, S5, S7), two MDR strains of E. coli (S2, S12),methicillin resistant MDR S. aureus (S4), and MDR S. typhimurium (S10)(FIGS. 5A-5B). CdSe-2.4 was consistent with data in Dh5α and showed nophotoeffect in clinical strains (FIGS. 6A-6B). CIS-1.9 showedphotoproliferation in two MDR strains of K. pneumoniae (S1, S5), MDR E.coli (S12), MDR P. aeruginosa (S6), and MDR A. baumannii (S13) (FIGS.7A-7B). Ag₂S-1.7, Cu₂S-2.1, and FeS-2.0 shown no significant photoeffect(FIGS. 8A, 8B, 9A, 9B, 10A, 10B).

Example 5: Potential Antimicrobial Therapeutic Application and SelectiveRedox Tuned Cell Death

In certain embodiments, the phototoxic response observed in CdTe-2.4allows for its use as a therapeutic agent for combating localized,bacterial infections. In other embodiments, QDs kill or preclude thegrowth of bacteria while leaving the surrounding host tissue healthy andintact. The present study addresses in part whether selective cell deathcan be observed due to QDs being tuned to specific organism-dependentcellular redox environments.

To evaluate antimicrobial potential, co-culture experiments wereperformed with E. coli and HEK293 cells. HEK293 cultures were grown intissue culture treated, 96-well plates for 24 hours to obtain 80%confluency, and then inoculated with E. coli and treated with quantumdots for 24 hours. To evaluate cell health, the HEK293 cells were fixedin 4% formaldehyde and dyed with the nuclear stain DAPI and actin stainPhalloidin Cruzfluor 488 conjugate to observe cell morphology. Prior tothe co-culture, E. coli was transformed to maintain a plasmidconstitutively expressing the mCherry fluorescent protein and wasobserved for cell density.

HEK293 cells, in absence of bacteria, did not exhibit a morphologicallyobservable photoeffect in the presence of CdTe-2.4, CdSe-2.4, or CIS-1.9(FIG. 18). The materials were also not inherently toxic to HEK293 at theQD concentrations studied. When co-cultured without QDs, there wascomparable growth of E. coli and consistent cell morphology of HEK293 inlight and dark. E. coli and HEK293 cells did not display a phototoxiceffect in the presence of CdSe-2.4, as predicted by the monocultures. Incontrast, CdTe-2.4 produced a phototoxic effect in E. coli and noobservable photoeffect in HEK293 cells, as predicted by monocultures(FIG. 11). This study showed that in co-culture selective cell death bythe survival of HEK293 cells and the eradication of E. coli cellsilluminated in the presence of CdTe-2.4 was obtained.

Example 6: Exploration of Other Metal Sulfides

To examine the potential of other nanoparticles made from distinctmetals, iron, silver, and copper sulfide were synthesized using ammoniumsulfide (FIG. 12). Based on STS measurements of these particles, FeSpossessed the nearest oxidation potential to the established requiredfor phototoxicity with a slight overpotential. Both Ag₂S and Cu₂S didnot align with any known photoeffect potential, and a corresponding lackof phototoxicity or photoproliferation was observed in all strainstested.

Example 7: Ligand Charge Effects

In one aspect, chemical instability of the QDs was evidenced by thechange in emission spectra (FIG. 22). In another aspect, colloidalinstability over time in relevant media was also observed. Withoutwishing to be limited by any theory, because the MPA ligands, whichelectrostatically stabilize the particles, remain unprotonated at pH 7.4this instability can be due to the desorption of the ligands through thereprotonation or dimerization of the thiol termini. In the first case,it may be possible to increase colloidal stability by replacing thenegatively-charged carboxylic acid ligands with an amine. This wouldcreate around the particle a layer of positive charge, which couldscreen incoming protons from being able to diffuse to the thiol group.CdTe with positive ligands (CdTe-CA) were obtained through ligandexchange of MPA coated dots. The initial stocks were filtered andre-dispersed in pH 6 medium containing excess cysteamine (CA) andallowed to react overnight.

The particles were initially well-dispersed, though the emission wasattenuated (FIG. 23A). The degradation profile of these particles werequantified in the same manner as the MPA-coated analogues (FIG. 23B).Instead of the two-regime curve shown previously, the CA coatedparticles exhibited only blue-shifting emission and a rapid loss ofemission intensity. After two hours of light exposure, thephotoluminescence was below the detection limit of the instrument, andthe population kept in dark were non-luminescent between four and fivehours. This is reflected in the absorbance spectra, which showed a rapidloss of colloidal stability with the increasing contributions ofscattering, and chemical instability through the loss of the primaryexcitionic feature. In certain embodiments, in biologically relevantmedia, positively charged ligands lead to lower stability overall thanthose coated with MPA.

From the observed growth curves the positively charged particles alsoexhibited much greater inherent toxicity than their negatively chargedcounterparts (FIGS. 23C-23D). Such an effect is also observed for otherstrains of bacteria and some mammalian cells. Without wishing to belimited by any theory, the positively charged CdTe-2.4 can more easilybind to the negatively charged cell membrane and thus be incorporatedwithin the cells at a higher concentration. Based on ICP analysis ofexposed cultures, the positive ligands are associated with the cells ina significantly higher concentration than the nominal cores (FIG. 23E).Thus, in certain embodiments, their poor stability and increasedinherent toxicity preclude such particles from being used within thepresent invention.

Example 8: ZnS Core Shells

One method of potentially increasing the chemical stability of the CdTequantum dots, while decreasing their inherent toxicity, would be to coatthem with a thin shell of a more biologically compatible material. Incertain embodiments, such construct is a type-I heterostructureconsisting of an emissive CdX core enveloped in a thin shell of ZnS,which protects the emission and decreases the overall toxicity. Withoutwishing to be limited by any theory, for therapeutic applications, theincreased stability with thicker shells is weighed against theincreasing tunneling barrier for moving photogenerated charges from thecore material to the intended targets. In certain embodiments, there isthe potential effect of different binding affinities between the coreand shell metals, which could possibly change the ability of theparticles to efficiently interact with the necessary species in themedium. Thus, ZnS shells with sub-monolayer thicknesses wereinvestigated.

CdTe particles were filtered to remove unreacted starting materials anddiluted to 2 μM in pH 11 water. Stock solutions containing zinc nitrateand thiourea (a sulfur source) were made and mixed in a 1:1 ratio withthe quantum dot stock, then allowed to react at 98° C. for 1 h. Thedeposition of the shell material was identified optically by changes inthe absorbance features and by red-shifting emission with greatercoverage (FIG. 24A). In certain non-limiting embodiments, addition ofthe zinc sulfide changes the electronic structure of the constituentparticles, without necessarily forming a true type-I junction. In othernon-limiting embodiments, zinc preferentially adds to the tellurium richfacets, and sulfur to the cadmium. Based on the bulk potentials, ZnTe'svalence band may fall within the nominal bandgap of CdTe as does CdS'sconduction band (FIG. 24B). In certain non-limiting embodiments, thefirst monolayers can alter the electronic structure of the core, eitherdue to defect formation in the low coverage regime, or due to thecreation of a closely aligned pseudo type-II junction approaching singlemonolayer deposition. Due to the high surface area to volume ratios insub-5 nm diameter particles, a plurality of atoms are involved with thecore-shell interface, thus having a significant effect on the hybridelectronic structure when a coating material is chosen whose individualinteractions with the core elements results in similar band positions.

Quantifying the elemental composition of the ZnS coated core shellsallows the calculation of the total surface coverage of the CdTe cores.A maximum coverage of about 25% Zn was obtained when the shell precursorsolutions contained zinc and sulfur at a concentration equivalent to twomono-layers equivalent (MLE) concentration relative to the cores. Usinghigher concentrations of ZnS resulted in the nucleation of thoseparticles directly, instead of thicker shell growth. The shell formationdoes not have an appreciable impact on the fluorescence quantum yield inrelation to the cores alone (FIG. 24A), indicating that any benefit dueto increased passivation is offset by the change in electronic structureat low loadings.

To test the stability of the core-shell structures relative to the coresan experiment was performed to track the degradation over time, andexhibited different degradation profiles (FIG. 24C). There was aninitial regime of slowly blue-shifting emission, which may represent theoxidation of the shell materials, where the addition of oxygen removessome of the trap-like states and moves the emission slightly closertowards the nominal cores. However, this effect was temporary, and oncethe shell was fully oxidized, similar red-shifting behavior as seen inthe cores begins to take place, followed by a rapid blue-shift andcollapse of the emission intensity. These measurements support theassertion that the core shell particles degrade significantly slowerthan the naked cores, lasting about twice as long, and that thepersistence of the emission implies that they are able to continuegenerating redox species as well.

To quantify the core shells' effects on bacterial inhibition they weredirectly compared to cores using the culture procedures describedelsewhere herein (FIGS. 25A-25B). The photoeffect was maintained withthe core shells, though there was a certain lag-time before they becomeeffective, as evidenced by the OD growth curves. There was nosignificant change in particle uptake relative to the cores, indicatingthat when in the same size regime the primary motivator for particleassociation is the capping ligand.

Example 9: Cd Overcoat

In certain embodiments, the observed oxidation occurs due to thereplacement of tellurium. In other embodiments, a strategy forincreasing stability is to overcoat the tellurium rich facets withadditional cadmium directly. The only effect this would have on theelectronic structure of the particles would be a slight decrease inbandgap due to the larger diameter, which is reflected in the consistentfeatures in the optical spectra (FIG. 26A). These particles weresynthesized in a similar manner as the ZnS core shells, only reactingfor 15 min. Elemental analysis of these particles indicates there was a30% increase in cadmium content compared to the cores, which translatesto 0.6 MLE coverage, showing complete passivation of the telluriumfacets. The increased passivation is reflected in the luminescencequantum yield of the overcoated particles, which is over twice that ofthe untreated cores.

In terms of stability, the cadmium overcoated samples consistentlyoutperformed both the cores and ZnS core shells (FIG. 26B). Unlike thoseother samples, the overcoated particles were still luminescent at 24 hof light illumination, and underwent much slower rates of degradationduring the first nine hours than the ZnS. The degradation curve alsoconsisted of a single monotonic phase of slow blue shifting, whichappears to be characteristic of this type of treated sample. In certainnon-limiting embodiments, as there are no exposed tellurium facets withwhich to readily exchange anions, degradation consists of only the slowdiffusion of oxygen through the cadmium layer.

When tested in vitro, the overcoat quantum dots retained a similar lagphase as the ZnS core shells, but had overall higher phototoxicity andan inherent toxicity which did not as strongly depend on particleconcentration (FIGS. 26C-26D). As the uptake of these particles did notsignificantly different than the untreated cores (FIG. 26E), theirenhanced stability and consistent efficacy indicate that thispassivation strategy improves the overall properties of therapeuticnanomaterials.

Example 10: Photoexcited Quantum Dot Functions in Combination withAntibiotics Against Multi Drug Resistant Gram-Negative Bacteria Methods

Bacterial Cell Culture:

Clinical isolates were growth in liquid cation adjusted Mueller Hintonbroth (CAMHB) (DIFCO) or on solid CAMHB and 1.5% agar. Clinical strainswere stored in 10% glycerol at −80° C. for long term storage. Replicateswere started from individual, single colonies off of solid plates andgrown for 16 h at 37° C. with 225 rpm shaking before beginningexperiments. Optical density was measured with a Tecan GENios at 562 nmwith a bandwidth of 35 nm.

IC₅₀ Measurement:

Overnight cultures of clinical isolates were diluted to a 0.5 McFarlandstandard in CAMHB with respective test concentration of antibiotic.Cultures were grown for 24 h at 37° C. with 225 rpm shaking. After 24 hof growth, Resazurin sodium salt (Sigma Aldrich) solution was added andthe reaction was monitored for fluorescence measuring every 5 min for 4h at 37° C. with 225 rpm shaking using 485/610 nm filters. The slope ofResazurin fluorescence was used a quantitative measure of cellmetabolism. The IC₅₀ was determined as the concentration of antibioticwhich caused a 50% reduction in slope compared to the same biologicalreplicate in no treatment.

Combinatorial Experiments:

Five antibiotic concentrations were selected for each strain, so thatthe levels tested would be below the IC₅₀, near the CLSI or definedbreakpoint, and near the IC₅₀. Concentrations of CdTe-2.4 were heldconstant for all strains at 12.5 nM, 25 nM, and 50 nM. Using thesemetrics, three biological replicates were tested from each strain with15 test conditions as well as monotherapy controls and a no treatmentcondition. Clinical strains were diluted 1:100 from overnight into testcondition and grown at 37° C. with 225 rpm. Optical density was measuredevery 30 min for the first 3 h and every hour subsequently until 8 h.After 8 h of growth, Resazurin sodium salt solution was added and thereaction was monitored for fluorescence measuring every 5 min for 4 h at37° C. with 225 rpm shaking using 485/610 nm filters. The slope ofResazurin fluorescence was used a quantitative measure of cellmetabolism.

Discussion

Multi-drug resistant (MDR) bacterial infections threaten the future ofour healthcare system due to pan-drug resistant bacteria. There is aneed for antimicrobial agents that are efficacious, either alone or incombination with current antibiotics, to treat MDR bacteria and mitigatethe antibiotic crisis. As demonstrated herein, compositions of theinvention can be used in combination with current antibiotics to inhibitMDR bacteria.

Cadmium telluride quantum dots with a band gap of 2.4 eV (CdTe-2.4) wereused in combination with other antibiotics to inhibit gram-negativeclinical isolates of MDR bacteria (FIGS. 22A-22B). Four gram-negativeclinical strains were used to investigate the ability of CdTe-2.4 tofunction as a therapeutic in combination with current antibiotics: a MDRstrain of Escherichia coli, a MDR strain of Salmonella typhimurium, anextended spectrum β-lactamase (ESBL) producing strain of Klebsiellapneumoniae, and a carbapenem resistant strain of E. coli. Theconcentration of antibiotic that inhibits 50% of the cultures growth(IC₅₀) was measured for the four strains against five antibiotics chosenfor their different mechanisms of action (FIG. 22B): a cephalosporinantibiotic (ceftriaxone), a fluoroquinone (ciprofloxacin), and threeprotein synthesis inhibitors (clindamycin and chloramphenicol, andstreptomycin).

MDR S. typhimurium and MDR E. coli were sensitive to chloramphenicol andMDR S. typhimurium was also sensitive to ciprofloxacin (FIG. 22B). WhenESBL K. pneumoniae was treated with 25 nM CdTe-2.4 and 8 μg/mLstreptomycin, significant inhibition of growth was observed, incomparison with no treatment and the corresponding monotherapies (FIG.22C). Addition of CdTe-2.4 allowed significant inhibition of growth ofESBL K. pneumoniae to a breakpoint amount of streptomycin that waspreviously ineffective.

A similar growth inhibition was observed in MDR S. typhimurium (FIG.22D). The degree of growth inhibition was dependent on the concentrationof CdTe-2.4 and the concentration of streptomycin (FIG. 22D).

The dual treatment against the MDR gram-negative bacteria significantlyreduced the number of viable cells in the culture in only 4 h oftreatment (FIGS. 28A-28B). Similar to MG1655, CdTe-2.4 generatedreactive oxygen species upon illumination with light in the multi-drugresistant strains, confirming the mechanism of action (FIG. 29).

The present studies have shown that multi-drug resistant bacteria can besuccessfully inhibited using a combination of antibiotic and tunedquantum dot or nanoparticle.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of promoting growth, killing, orpreventing or hampering growth, of a first cell, the method comprisingirradiating the first cell with radiation ranging from about 400 nm toabout 1,000 nm in the presence of at least one semiconductor-containingnanoparticle with a band edge redox potential, under conditions wherebythe at least one nanoparticle penetrates the first cell, whereby growthof the first cell is promoted, growth of the first cell is prevented orhampered, or killing of the first cell is promoted.
 2. The method ofclaim 1, wherein the nanoparticle comprises a quantum dot (QD).
 3. Themethod of claim 1, wherein the first cell is irradiated with radiationranging from about 750 nm to about 1,000 nm.
 4. The method of claim 1,wherein the first cell is a bacterium.
 5. The method of claim 4, whereinthe bacterium comprises at least one selected from the group consistingof K. pneumonia, E. coli, S. aureus, P. aeruginosa, A. baumannii and S.typhimurium.
 6. The method of claim 4, wherein the first cell comprisesa gram-negative bacterium, and wherein the first cell is furthercontacted with at least one gram-negative antibacterial agent.
 7. Themethod of claim 6, wherein the at least one antibacterial agent is acephalosporin antibiotic, fluoroquinone, or protein synthesis inhibitor.8. The method of claim 6, wherein the at least one antibacterial agentis selected from the group consisting of Amikacin, Aztreonam, Cefdinir,Cefaclor, Cefamandole, Cefditoren, Cefixime, Cefoperazone, Cefotaxime,Cefoxitin, Cefpodoxime, Cefprozil, Cefuroxime, Ceftazidime, Ceftibuten,Ceftobiprole, Ceftriaxone, Chloramphenicol, Ciprofloxacin, Clindamycin,Colistin, Ertapenem, Doripenem, Gatifloxacin, Gentamicin,Imipenem/Cilastatin, Kanamycin, Levofloxacin, Meropenem, Metronidazole,Moxifloxacin, Neomycin, Netilmicin, Ofloxacin, Paromomycin, Polymyxin B,Streptomycin, Thiamphenicol, Tigecycline, and Tobramycin.
 9. The methodof claim 6, wherein the concentration or amount of the antibacterialagent that is required to kill, or prevent or hamper the growth of, thefirst cell in the presence of the at least one nanoparticle is lowerthan the concentration or amount of the antibacterial agent that isrequired to kill, or prevent or hamper the growth of, the first cellwhen the antibacterial agent is used in the absence of the at least onenanoparticle.
 10. The method of claim 1, wherein the first cell is inthe presence of a second cell, and wherein irradiation of the first andsecond cells in the presence of the at least one nanoparticle has nomeasurable effect on the growth, metabolism or survival of the secondcell.
 11. The method of claim 10, wherein the second cell is mammalian.12. The method of claim 2, (i) wherein the QD comprises CuInS₂, andwherein growth of the first cell is promoted or (ii) wherein the QDcomprises CdTe, and wherein growth of the first cell is prevented orhampered, or killing of the first cell is promoted.
 13. The method ofclaim 2, wherein the QD comprises CdTe, and wherein growth of the firstcell is prevented or hampered, or killing of the first cell is promoted.14. The method of claim 2, wherein (i) growth of the first cell ispromoted, and wherein the band edge of the conduction band state(reduction potential) of the QD is about +0.2 V and the band edge of thevalence band state (oxidation potential) of the QD is about −1.8 V, asreferenced to NHE (standard hydrogen electrode), or (ii) growth of thefirst cell is prevented or hampered, or killing of the first cell ispromoted, and wherein the band edge of the conduction band state(reduction potential) of the QD is about +0.35 V and the band edge ofthe valence band state (oxidation potential) of the QD is about −2.1 V,as referenced to NHE (standard hydrogen electrode).
 15. The method ofclaim 2, wherein the at least one nanoparticle comprises CdTe, andwherein the at least one nanoparticle is at least partially coated withat least one selected from the group consisting of ZnS and CdS.
 16. Themethod of claim 1, wherein irradiation has at least one effect selectedfrom the group consisting of: changing redox homeostasis in the firstcell, and generating at least one light-activated reactive species inthe first cell.
 17. A method of altering redox homeostasis in a cell,the method comprising irradiating the cell with radiation ranging fromabout 400 nm to about 1,000 nm in the presence of at least onesemiconductor-containing nanoparticle with a band edge redox potential,under conditions whereby the at least one nanoparticle penetrates thecell, whereby redox homeostasis in the cell is altered.
 18. The methodof claim 17, wherein the at least one nanoparticle comprises a quantumdot (QD).
 19. The method of claim 17, wherein the cell is irradiatedwith radiation ranging from about 750 nm to about 1,000 nm.